1. Field of the Invention
The present invention relates to specimen holder, specimen inspection apparatus, and specimen inspection method permitting a specimen consisting of cultured cells or the like to be observed or inspected. The present invention also relates to a method of fabricating the specimen holder.
2. Description of Related Art
In life science and pharmaceutical applications, it is important that stimuli (such as electricity, chemical substances, and medicines) are given to biological cells and that resulting reactions are observed. In the past, optical microscopes have been used for such observations. Manipulators have been used to give stimuli to the cells. However, important parts to be observed are frequently microscopic regions of less than 0.1 μm, which cannot be observed with optical microscopes.
For example, diseases arising from inability to exchange substances among biological cells normally include hypertension, diabetes insipidus, arrhythmia, muscular disorders, diabetes, and depression. Exchange of substances among cells is performed by ion channels having sizes of about 10 nm and existing in cell membranes. Because it is difficult to observe such ion channels with optical microscopes, there has been a demand for a technique enabling observation using a scanning electron microscope (SEM) having high resolution.
However, a specimen to be inspected with an inspection apparatus incorporating SEM capabilities is normally placed in a specimen chamber whose inside pressure has been reduced by vacuum pumping. The specimen placed in the specimen chamber, which, in turn, is placed in a reduced-pressure ambient in this way, is irradiated with an electron beam (charged-particle beam). Secondary signals, such as secondary electrons or backscattered electrons, produced from the specimen in response to the irradiation are detected.
In such inspection of a specimen using SEM, the specimen is exposed to a reduced-pressure ambient. Therefore, moisture evaporates from the specimen, so that the cells die. It has been impossible to observe reactions of living cells to a stimulus.
Accordingly, when an inspection is performed under the condition where the specimen contains moisture, it is necessary to prevent the specimen from being exposed to the reduced-pressure ambient; otherwise, moisture would evaporate from the specimen. One conceivable method of inspecting a specimen using SEM without exposing the specimen to a reduced-pressure ambient in this way consists of preparing a specimen holder (specimen capsule) whose opening (aperture) has been sealed off by a film, placing the specimen in the holder, and installing the holder in an SEM specimen chamber that is placed in the reduced-pressure chamber.
The inside of the specimen holder in which the specimen is placed is not evacuated. The film that covers the opening formed in the specimen holder (specimen capsule) can withstand the pressure difference between the reduced-pressure ambient inside the SEM specimen chamber and the ambient (e.g., atmospheric-pressure ambient) of the inside of the specimen holder that is not pumped down. Furthermore, the film permits an electron beam to pass therethrough (see JP-T-2004-515049).
When a specimen is inspected, an electron beam is directed at the specimen within the specimen capsule from outside the capsule via the film on the capsule placed in the SEM specimen chamber that is in the reduced-pressure ambient. Backscattered electrons are produced from the irradiated specimen. The backscattered electrons pass through the film on the capsule and are detected by a backscattered electron detector mounted in the SEM specimen chamber. Consequently, an SEM image is derived.
However, with this technique, the specimen is sealed in the closed space and so it has been impossible to give a stimulus to cells using a manipulator. Furthermore, where a specimen consisting of cells is sealed in the specimen capsule and then the cells are observed or inspected in vitro, there arises a problem.
In particular, the cells are previously adsorbed onto a laboratory dish. A culture medium is put over the cells. The cells are cultured in an ambient having a temperature of 36°-38° C. (normally 37° C.) and a carbon dioxide concentration of 3% to 10% (normally 5%). When the cells are observed, the cells are harvested from the dish and subcultured into the specimen capsule. However, the environment inside the specimen capsule is different from the environment inside the dish. Therefore, the possibility that the cells survive in the specimen holder (specimen capsule) is low. Furthermore, in the specimen capsule described in the above-cited JP-T-2004-515049, it is possible to put only about 15 μl of medium into the capsule. Because the ambient environments including pH and osmotic pressure vary in a short time, it has been difficult to culture the cells.
An example of a method of obtaining an SEM image by preparing a film withstanding the pressure difference between vacuum and atmospheric pressure, irradiating a specimen with an electron beam via the film, and detecting backscattered electrons produced from the specimen in this way is described also in Atmospheric scanning electron microscopy, Green, Evan Drake Harriman, Ph.D., Stanford University, 1993 (especially, Chapter 1: Introduction).
Examples in which two films of the structure described above are placed opposite to each other with a specimen interposed between the films and an image is acquired by a transmission electron microscope are described in JP-A-47-24961 and JP-A-6-318445. Especially, JP-A-47-24961 also states a case in which an SEM image of the specimen interposed between such films is acquired.
Morphological variations based on reactions of cells after a stimulus is given to the cells using a manipulator take place in microscopic regions within the cells. Therefore, the variations cannot be observed with an optical microscope. Hence, observation using SEM is essential. In order to observe the cells by SEM while maintaining the liquid, the specimen (cells) cultured on a laboratory dish is sealed in a specimen capsule. An electron beam is directed at the specimen via a film formed on the capsule, thus obtaining an image. However, the inside of the specimen capsule is a closed space. Consequently, it has been impossible to use a manipulator for giving a stimulus. Furthermore, the possibility that cells sealed in the capsule survive has been low.